凝血酶调节蛋白植物凝集素样片段的原核表达及抗体制备

The expression of the lectin-like domain of thrombomodulin and production of anti-thrombomodulin antibodies

目的 :制备凝血酶调节蛋白 (thrombomodulin ,TM)抗体。方法 :原核表达目的蛋白 ,将表达的融合蛋白纯化后免疫小鼠。以EVC30 4包板的cell ELISA检测抗体滴度 ,用Westernblot和免疫组化鉴定抗体特异性。结果 :目的蛋白在大肠杆菌中大量表达形成包涵体 ,将表达蛋白纯化并复性后免疫小鼠 ,3次免疫后得抗体效价为 1∶4 0 0 0 ,经加强免疫后抗体效价达到1∶80 0 0 ;特异性较好。结论 :在天然TM提取纯化困难的情况下 。

Objective:To produce anti-thrombomodulin antibodies.Methods:The mice were immuned with the protein expressed and purified from E.coli.M15.The production of anti-TM have been detected.Results:The expression of target protein at high levels results in the formation of inclusion bodies.The expressed protein was purified by Ni-NT agarose and was refolded.Specific anti-sera with a titer of 4 000 have been obtained after the mouse received 3 times immunization and a titer of 8 000 with 4th immunization detected by cell-ELISA coated with EVC-304.Specificity has been identified by Western blot and immunohistochemistry.Conclusion:The production of antibody through immunization with the expressed protein is a feasible method due to the difficulties in obtaining and purification of natural thrombomodulin